Simultaneous Quantification of Vibrio metoecus and Vibrio cholerae with Its O1 Serogroup and Toxigenic Subpopulations in Environmental Reservoirs

Abstract

Vibrio metoecus is a recently described aquatic bacterium and opportunistic pathogen, closely related to and often coexisting with Vibrio cholerae. To study the relative abundance and population dynamics of both species in aquatic environments of cholera-endemic and cholera-free regions, we developed a multiplex qPCR assay allowing simultaneous quantification of total V. metoecus and V. cholerae (including toxigenic and O1 serogroup) cells. The presence of V. metoecus was restricted to samples from regions that are not endemic for cholera, where it was found at 20% of the abundance of V. cholerae. In this environment, non-toxigenic O1 serogroup V. cholerae represents almost one-fifth of the total V. cholerae population. In contrast, toxigenic O1 serogroup V. cholerae was also present in low abundance on the coast of cholera-endemic regions, but sustained in relatively high proportions throughout the year in inland waters. The majority of cells from both Vibrio species were recovered from particles rather than free-living, indicating a potential preference for attached versus planktonic lifestyles. This research further elucidates the population dynamics underpinning V. cholerae and its closest relative in cholera-endemic and non-endemic regions through culture-independent quantification from environmental samples.

Keywords: O1; Vibrio cholerae; Vibrio metoecus; cholera-endemic; qPCR; serogroup; toxigenic and non-toxigenic.

Figure 1

Sampling sites for environmental water samples collected to evaluate the qPCR assay developed in this study. (A) Oyster Pond (Falmouth, MA, USA) a non-endemic site for cholera. (B) Map of Bangladesh, identifying the two coastal regions (Kuakata and Mathbaria) and inland region (Dhaka) where samples were collected.

Figure 2

Multiplex real-time qPCR for simultaneous detection and quantification of V. cholerae and V. metoecus. Four gene markers with fluorogenic probes were used: viuB (V. cholerae specific), ctxA (toxigenic V. cholerae specific), rfbO1 (V. cholerae O1 specific) and mcp (V. metoecus specific). Template DNA was purified from reference cultures (V. cholerae N16961 and V. metoecus RC341) and was serially diluted in 10-fold increments to yield concentration ranging from 3 × 10⁶ to 3 copies per reaction (from left to right). Fluorescence was measured in relative units. The panel (A) illustrates amplification curves and the panel (B) shows their corresponding standard curves. Each reaction was done in triplicate.

Figure 3

Temporal variation of abundance for V. cholerae along with its toxigenic and serogroup O1 subpopulations and its close relative V. metoecus in Oyster Pond, MA, USA. Environmental water samples collected during the months of June to August in two successive years: 2008 (A) and 2009 (B), were analyzed using the developed qPCR assay. The viuB gene was used to quantify total V. cholerae; ctxA and rfbO1 were used to measure toxigenic V. cholerae and V. cholerae O1, respectively; the abundance of V. metoecus was estimated using the mcp gene. All four genes were tested for each sample and the absence of a bar in the graph denotes that the target gene was absent or present below the detection limit of the assay. Each qPCR reaction was run in triplicate. Mean values are shown with error bars indicating the standard deviation between three technical replicates. ctxA could not be detected at any time sampled at this site.

Figure 4

Distribution in different water fraction sizes of V. cholerae along with its toxigenic and serogroup O1 subpopulations and its close relative V. metoecus in Oyster Pond, MA, USA. Environmental water samples were collected during the months of June (A), July (B) August (C) and September (D) in 2009. These samples were fractionated by size through sequential filtration and bacteria were quantified by qPCR of marker genes on DNA extracted from the filters. The viuB gene was used to quantify total V. cholerae; ctxA and rfbO1 were used to measure toxigenic V. cholerae and V. cholerae O1, respectively; the abundance of V. metoecus was quantified using the mcp gene. Each qPCR reaction was run in triplicate. Mean values are shown with error bars indicating the standard deviation between technical replicates. Temperature, pH and salinity of water collected each month are shown in boxes on the upper right corner of each graph. ND indicates not done.

Figure 5

Abundance of V. cholerae along with its toxigenic and serogroup O1 subpopulations and its close relative V. metoecus in two different coastal regions in Bangladesh. Environmental water samples were collected from Kuakata and Mathbaria during the month of May in 2014 and bacteria were quantified by qPCR of marker genes. The viuB gene was used to quantify total V. cholerae; ctxA and rfbO1 were used to measure toxigenic V. cholerae and V. cholerae O1, respectively; the abundance of V. metoecus was quantified using the mcp gene. Each qPCR reaction was run in triplicate. Mean values are shown with error bars indicating the standard deviation between technical replicates.

Figure 6

Abundance of V. cholerae along with its toxigenic and serogroup O1 subpopulations and its close relative V. metoecus in two different coastal regions in Bangladesh. Environmental water samples were collected from Kuakata (A) and Mathbaria (B) during the month of May in 2014 and bacteria were quantified by qPCR of marker genes. The viuB gene was used to quantify total V. cholerae; ctxA and rfbO1 were used to measure toxigenic V. cholerae and V. cholerae O1, respectively; the abundance of V. metoecus was quantified using the mcp gene. Each qPCR reaction was run in triplicate. Mean values are shown with error bars indicating the standard deviation between technical replicates.

Figure 7

Temporal variation of abundance for V. cholerae along with its toxigenic and serogroup O1 subpopulations and its close relative V. metoecus in a central region of Bangladesh (Dhaka). Environmental water samples were collected bi-weekly from the months of October 2015 to March 2016 and bacteria were quantified by qPCR of marker genes. The viuB gene was used to quantify total V. cholerae; ctxA and rfbO1 were used to measure toxigenic V. cholerae and V. cholerae O1, respectively; the abundance of V. metoecus was quantified using the mcp gene. Each qPCR reaction was run in triplicate. Mean values are shown with error bars indicating the standard deviation between technical replicates.